Apoptosis is responsible for the functions of removing cells that become no longer necessary during development and abnormal cells, of homeostasis, and of body defense reactions to remove injured cells, and its mechanisms have been elucidated gradually at the molecular level. Such molecular abnormalities and the collapse of the control mechanism damage the physiological functions of apoptosis, and result in causal factors or aggravation factors for various diseases. For example, excessively suppressed apoptosis leads to abnormal proliferation of cells to be removed originally, which in turn induces tumoral diseases, autoimmune diseases and the like. Conversely, abnormally promoted apoptosis leads to the death of cells that must exist originally, which in turn causes neurodegenerative diseases and the like.
The mitogen-activated protein (MAP) kinase cascade is a signal transduction mechanism through which MAP kinase kinase kinase (MAPKKK) activated by a physicochemical stress or an inflammatory cytokine such as tumor necrosis factor-α (TNF-α) or interleukin-1 (IL-1) sequentially activates MAP kinase kinase (MAPKK) and MAP kinase (MAPK), and cells, in response to these stimuli, exhibit phenotypes such as survival, proliferation, differentiation and death (apoptosis). c-Jun N-terminal kinase (JNK) and p38 MAP kinase (p38) are known as MAPKs that play a part of the role in the signal transduction pathway to induce apoptosis (see, for example, Science, 270, 1326 (1995)). Furthermore, they are also involved in the evoking of inflammatory reactions by inducing the production of inflammatory cytokines.
JNK and p38 are activated by MKK4/7 and MKK3/6, respectively, which are MAPKKs. These MAPKKs are activated by single MAPKKK known as apoptosis signal-regulating kinase 1 (ASK1) (JP 10-93. A, Science, 275, 90-94 (1997)). In addition to ASK1, many MAPKKKs have been reported, and ASK1 is characterized by the capability of inducing apoptosis of cells through signal transduction via the activation of JNK and/or p38. Recently, ASK1 activation has been suggested to be involved in cell differentiation such as keratinocyte differentiation and PC12 cell axon elongation as well, and ASK1 has been shown to play an important role, not only in apoptosis, but also in the control of cell fate. Furthermore, ASK1 has also been shown to be involved in the evoking of inflammatory reactions by inducing the production of inflammatory cytokines.
Because ASK1 is an important molecule that influences the subsequent fate of cells as described above, its activation is considered to be involved by various factors and undergoes complicated control. To date, it has been reported that the formation of a homo-oligomer by ASK1 molecules and the subsequent phosphorylation of threonine in the activation loop are essential to the activation of ASK1, and the phosphorylation is based mainly on auto-phosphorylation by ASK1, but the presence of another kinase has been suggested (Journal of Cellular Physiology, 191, 95-104 (2002)). On the other hand, protein phosphatase 5 (PP5) is considered to restore activated ASK1 to an inactivated state by binding directly to ASK1 under stimulation with H2O2 and dephosphorylating threonine (EMBO Journal, 20, 6028-6036 (2001)). Furthermore, it has also been reported that thioredoxin, a redox control factor, acts as an ASK1 activation inhibitor as constitutively bound to the N-terminal domain of ASK1 in the absence of oxidation stress and leaves ASK1 upon exposure to oxidation stress and hence causes the activation of ASK1 (EMBO Journal, 17, 2596-2606 (1998)), that during the activation of ASK1 by TNF-α, TNF receptor-associated factor 2 (TRAF2) binds to the C-terminal domain of ASK1 to cause the activation of ASK1 (Molecular Cell, 2, 389-395 (1998)), and that the 14-3-3 protein inhibits the activation of ASK1 by binding to the C-terminal domain (Proceedings of National Academy of Sciences, USA, 96, 8511-8515 (1999)).
Because treating cells of an ASK1 knockout mouse with an endoplasmic reticulum stress inducer significantly suppresses apoptosis compared to cells of a wild-type mouse, it is suggested that ASK1 is closely associated with the induction of apoptosis by endoplasmic reticulum stress, and that the above-described ASK1 inhibitors such as thioredoxin and the 14-3-3 protein, ASK1 dominant negative variants, ASK1 antisense oligonucleotides and the like are effective in the prophylaxis or treatment of diseases associated with endoplasmic reticulum stress, such as neurodegenerative diseases (e.g., polyglutamine disease and the like) (WO 02/38179). Nishitoh et al. showed that the accumulation of an abnormal protein in polyglutamine disease evokes endoplasmic reticulum stress, hence leads to the formation of a tripartite complex of the stress sensor molecule IRE1, TRAF2 and ASK1 and activates ASK1, and induces apoptosis (=nerve cell death) via the activation of JNK (Genes Development, 16, 1345-1355 (2002)).
Although the physiological importance of ASK1 and its association with disease have been elucidated gradually as described above, much remains unknown about the mechanisms of ASK1 activation control and apoptosis induction/inflammatory reaction evoking via ASK1, due partially to the complexity thereof, and there is a demand for further advances in the relevant research.
Accordingly, the present invention is directed to provide new findings on the mechanisms of activation of ASK1 and the mechanisms of apoptosis induction/inflammatory reaction evoking mediated thereby. That is, it is an object of the present invention to identify a novel ASK1-binding protein that has not been known to date, and to elucidate how ASK1 activation is regulated by the protein. It is another object of the present invention to provide a novel prophylactic or therapeutic means for various diseases involved by ASK1, on the basis of the interaction of the protein and ASK1.